Herpes Simplex Virus (HSV) is a major pathogen responsible for oral, genital and sight-threatening ocular infections which can be life threatening in immunocompetent adults and even more severe in individuals with compromised immune systems. Production of concatemeric DNA is an essential step for the generation of progeny virus as the packaging machinery must recognize longer-than-unit-length concatemers during encapsidation; however, the mechanism by which they are formed is very poorly understood. Although it has been proposed that the viral genome circularizes and rolling circle replication leads to the formation of concatemers, several lines of evidence suggest that HSV DNA replication is more complex and may involve recombination-dependent replication reminiscent of bacteriophages lambda and T4. We have previously shown that HSV encodes a two-subunit viral recombinase (UL12 and ICP8). Furthermore cellular DNA Damage Response (DDR) proteins have been shown by us and others to both positively and negatively influence the production of infectious progeny virus. The two subunit viral recombinase (UL12 and ICP8) interacts with several DDR proteins. It is now clear that DDR proteins function in at least four pathways, three that require some amount of homology (A-NHEJ, HR and SSA) and one that does not (C-NHEJ). We hypothesize that HSV navigates this complex environment to ensure the production of viral genomes that can be packaged into infectious virus. The central hypothesis is that HSV infection requires the activation of one or more of the homology dependent repair/recombination pathways and that viral proteins including the immediate early E3 ubiquitin ligase ICP0 and the viral recombinase (UL12 and ICP8) act to influence pathway choice. Furthermore we suggest that the C-NHEJ pathway is inactivated by viral proteins in order to promote end resection and the homology dependent pathways leading to the formation of concatemers that can be processed into infectious virus. In aim 1, we will determine which cellular pathways are activated and inactivated by HSV infection and the consequences of these actions. In Aim 2 we will test the hypothesis that UL12 affects DDR signaling and directs pathway choice. In aim 3 we will test the hypothesis that the structure of viral DNA depends on the repair pathway activated during infection and that in the absence of UL12, inappropriate pathway choice leads to the accumulation of structurally aberrant DNA. It is expected that this study will improve our understanding of the mechanism of viral DNA replication, provide information on the functions of cellular proteins important for genome stability and cancer biology and aid in the development of new antiviral therapies. PUBLIC HEALTH RELEVANCE: Herpes Simplex Virus Types 1 and 2 (HSV-1 and HSV-2), are ubiquitous human pathogens responsible for oral, genital and sight-threatening ocular infections that can be life threatening even in immunocompetent adults. In individuals with compromised immune systems, HSV infections are often fatal. Our goal is to contribute to the understanding of mechanisms underlying replication of this virus and to provide information that will lead to novel and effective antiviral therapies.